Methods and apparatus for the isolation and enrichment of circulating tumor cells

ABSTRACT

Embodiments in accordance with the present invention relate to methods and apparatuses for concentrating and isolating Circulating Tumor Cells (CTCs) from body fluids. One embodiment of the present invention includes a micro-fabricated or nano-fabricated device having channels configured for separating and excluding. Embodiments in accordance with the present invention utilize features that reduce the hydrodynamic pressure experienced by the cells during the separation, isolation and concentration processes, and therefore reduce the likelihood of cell lysis or other damage to the cells.

CROSS-REFERENCE TO RELATED APPLICATION

The instant nonprovisional patent application is a continuation-in-partof U.S. patent application Ser. No. 11/202,416 filed Aug. 11, 2005 andincorporated by reference in its entirety herein for all purposes.

GOVERNMENT FUNDING

The subject matter described herein was made with U.S. Governmentsupport under National Institute of Health (NIH) Grant Number R01GM65293 and the Puget Sound Partners for Global Health Pilot Project(PSPGH). The United States Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Body fluid is a complex mixture of different cell types and biologicalparticles. Blood, for example, includes plasma and cells (red bloodcells, white blood cells, platelets) and the cells occupy about 55% ofblood. Plasma is mostly water and it transfers proteins, ions, vitamins,enzymes, hormone, and other chemicals to cells in the body. Red bloodcells are about 6 to 8 μm in size and serve to provide oxygen to cells.White blood cells are about 10 to 13 μm in diameter and they defend thebody from disease as a part of an immune system by fighting againstforeign virus and bacteria. Platelets are the smallest cells, 1.5 to 3μm, and they stop bleeding by forming blood clots. Fluids in addition toblood, such as saliva, tear, urine, cerebral spinal fluid as well asother body fluids in contact with various organs (e.g. lung) containmixtures of cells and bioparticles.

The type and amount of cells and bioparticles that are present in aparticular body fluid (e.g. blood) includes information about the healthof the organism, and in the case of an infected individual, informationabout the diagnosis and prognosis of the disease. For example, anemiacan be diagnosed by counting the number of red blood cells within a unitvolume of blood. Similarly, elevated white blood cell count is astandard screen for signs of heightened immune response, which is oftendue to infection.

In diseases such as HIV, the level of CD4+ T-lymphocytes (CD4+ T-cells)in blood indicates the degree of disease progression. In fact, the CDCPublic Health Service recommends monitoring the level of CD4+ T-cellsevery 3-6 months in all HIV-infected persons as a way to initiateappropriate treatment strategies. Another example is malaria diagnosis,in which the number of parasitized erythrocytes among normalerythrocytes and leucocytes is counted. Yet another example is in cancerdiagnosis and prognosis—tumor cells can exfoliate from solid tumors andtransport throughout the body via the blood stream or other body fluids(e.g. lung cancer cells may exfoliate into the fluid in contact with thelung and prostate cancer cells into urine). These circulating tumorcells are present in extremely low concentrations, and their isolationand detection among the other cells present in the fluid is required fordiagnosis and prognosis.

BRIEF SUMMARY OF THE INVENTION

Embodiments in accordance with the present invention relate to methodsand apparatuses for concentrating and isolating Circulating Tumor Cells(CTCs) from body fluids. One embodiment of the present inventionincludes a micro-fabricated or nano-fabricated device having channelsconfigured for separating and excluding. Embodiments in accordance withthe present invention utilize features that reduce the hydrodynamicpressure experienced by the cells during the separation, isolation andconcentration processes, and therefore reduce the likelihood of celllysis or other damage to the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a zero-dimensional channel with a target particle.

FIG. 1B illustrates a one-dimensional channel with a target particle.

FIG. 1C illustrates an aperture of rectangular configuration.

FIG. 1D illustrates an arbitrary object.

FIGS. 1E and 1F illustrate concave apertures.

FIGS. 1G, 1H, and 1J illustrate apertures.

FIG. 2A illustrates a substrate having an axial flow configuration.

FIG. 2B illustrates a substrate having a radial flow configuration.

FIGS. 3A and 3B illustrate a substrate in the form of a membraneconfigured for axial flow.

FIGS. 4A-4G illustrate various aperture cross sections.

FIGS. 5A-5D illustrate a sequence for fabricating an exemplary device.

FIGS. 6A-6D illustrate a sequence for fabricating an exemplary device.

FIG. 7 illustrates an injection molding system for fabricating andreplicating an exemplary device.

FIG. 8 illustrates scanning electron microscopy images of an exemplarydevice.

FIGS. 9A-9F illustrate the cell separation and enrichment operations ofan exemplary device.

FIGS. 10A and 10B illustrate top and side elevation views of azero-dimension channel.

FIGS. 10C-10H illustrate a cell lysing event by flowing a biofluidcontaining cells through a zero-dimensional channel.

FIGS. 11A-11C illustrate various scenarios that a biological cell orparticle can experience pressure forces.

FIG. 12 illustrates the pressure distribution around a free sphericalparticle in a flow.

FIG. 13 illustrates the pressure experienced by a cell lodged at aseparation channel entrance as a function of the percentage of channelcross-sectional area blocked.

FIG. 14A illustrates cellular membrane deformation as a result of a celllodged at a pore subject to applied pressure differential.

FIG. 14B illustrates cellular membrane deformation as a result of a celllodged at two pores subject to applied pressure differential.

FIGS. 15A and 15B illustrate modeled views of channels with targetparticles.

FIGS. 16A-16F illustrate trapping of malaria-infected red blood cells(RBC) and white blood cells (WBC).

FIG. 17 illustrates trapping of a cancer cell by an exemplary device.

FIG. 18 illustrates a flow channel with examples of relief patterns(topographical features) on the bottom wall of the channel.

FIG. 19 illustrates a production process of an exemplary device usingdirect etching.

FIG. 20 illustrates a production process of an exemplary device usingpolymer resin to replicate from a master mold.

FIGS. 21A and 21B illustrate the preparation of tumor cell samples byusing a micropipette to pick up individual tumor cells (arrow).

FIG. 22A illustrates the percent of breast cancer cells (MCF-7)recovered using an exemplary device (indicated with ♦).

FIG. 22B illustrates a still image of a breast cancer cell (arrow)moving through an exemplary device.

FIG. 23A illustrates the percent of breast cancer cells recovered usingan exemplary device in a sample mixed with human whole blood.

FIG. 23B illustrates the breast cancer cells isolated using an exemplarydevice.

FIG. 23C illustrates the fluorescent image of FIG. 23B to confirm thatthe cells isolated were indeed cancer cells.

FIG. 24 illustrates the percent of lung cancer cells (A549, indicatedwith ♦) recovered using an exemplary device.

FIG. 25 illustrates the percent of colorectal cancer cells (HT-29,indicated with ♦) recovered using an exemplary device.

FIG. 26 shows a size distribution of cancer cells from lung (A549) andbreast (MCF-7) cell lines.

FIG. 27 shows a recovery rate of cancer cells from whole human bloodspiked with breast cancer cells.

FIG. 28 shows a percentage of cancer cell recovered when the exclusioncondition (the relative ratio of cell diameter and the channel width) isaltered.

FIGS. 29A-B show an example of effusive filtration featuringconstriction of the feed chute.

FIGS. 30A-B show an example of effusive filtration featuring a180-degree bend.

FIGS. 31A-B show an example of effusive filtration featuring anexpansion of flow channel.

DETAILED DESCRIPTION OF THE INVENTION

This document pertains generally to methods and systems for isolating,separating, and concentrating biological cells and biological particlesfrom complex biofluid mixtures and, in particular but not by way oflimitation, to methods and systems with features to reduce cell lysis orcellular membrane damage during cell and particle isolation andseparation.

The following detailed description includes references to theaccompanying drawings, which form a part of the detailed description.The drawings show, by way of illustration, specific embodiments in whichthe invention may be practiced. These embodiments, which are alsoreferred to herein as “examples,” are described in enough detail toenable those skilled in the art to practice the invention. Theembodiments may be combined, other embodiments may be utilized, orstructural, logical and electrical changes may be made without departingfrom the scope of the present invention. The following detaileddescription is, therefore, not to be taken in a limiting sense, and thescope of the present invention is defined by the appended claims andtheir equivalents.

In this document, the terms “a” or “an” are used, as is common in patentdocuments, to include one or more than one. In this document, the term“or” is used to refer to a nonexclusive or, unless otherwise indicated.Furthermore, all publications, patents, and patent documents referred toin this document are incorporated by reference herein in their entirety,as though individually incorporated by reference. In the event ofinconsistent usages between this document and those documents soincorporated by reference, the usage in the incorporated reference(s)should be considered supplementary to that of this document; forirreconcilable inconsistencies, the usage in this document controls.

Biological cells are often sensitive to local pressure change becausecellular membranes are not rigid. In filtering or isolating cells bymechanical exclusion, exposure of the cells to a high pressureenvironment can cause lysing. Lysis refers to the disintegration,rupturing or destruction of a cell or bacteria. With a cell, such abreakdown is caused by damage to the plasma (outer) membrane andsubsequent loss of cell contents (cytoplasm, organelles or nucleus)resulting from physical insult to the cell. The present subject matterreduces the incidence of lysis in separation, concentration, filtrationor isolation.

The present subject matter relates to a micro-fabricated andnano-fabricated device and method useful for separating, concentratingand isolating microscopic or nanoscopic objects such as biologicalcells, macromolecules, colloidal particles, particulates, or micro-beadsand nano-beads using an array of one-dimensional channels. The devicemay be used to isolate, purify, and concentrate a subpopulation ofbiological cells to facilitate clinical diagnosis of diseases such asmalaria, AIDS and cancer.

As used herein, a fluid can include a liquid or a gas and an object ortarget particle can include a cell, a bacteria, a virus, a biologicalnano-particulate, a biological micro-particulate or other object. Thetarget particle can include an organic or inorganic object.

FIGS. 1A and 1B illustrate sample geometry for mechanically excluding acell, particulate or other immiscible objects from the immersed carrierfluid. FIG. 1A illustrates a geometric configuration where circularchannel 20 is used to exclude target particle 35. In this figure, targetparticle 35 is disposed near lumen 25 of channel 20 and arrow 30 denotesthe fluid flow direction, which is a manifestation of a pressuredifferential between an input side and an output side of channel 20.Since the approximate outside diameter of target particle 35 is largerthan the diameter of lumen 25, with the fluid flow traversing along thechannel as shown by arrow 30, lumen 25 is occluded. When occluded, fluidflow in channel 20 ceases. With continued application of the pressuredifferential, such as for example, in an attempt to re-establish theflow, under some circumstances, target particle 35 will distort andpossibly rupture.

Channel 20 is shown to have a circular cross section and is sometimesreferred to as a zero-dimensional channel. A zero-dimensional channelhas a pore with a diameter (or mean diameter in the case of filtersbased on cross-linked or highly branched matrices) smaller than thedimensions of the object intended to block, and thus mechanicallyprevents an object of diameter larger than that of the channel fromtraversing through the channel. The entrance of the channel becomesenriched with the excluded object and the fluid passing through thechannel, which is sometimes referred to as the filtrate, becomes devoidof the excluded object, thus accomplishing isolation of the object,separation of the object from the filtrate, and concentration of theobject near the entrance.

FIG. 1B illustrates channel 100A having a cross-sectional geometryaccording to the present subject matter which also provides mechanicalexclusion of target particle 35 in fluid flow direction indicated byarrow 110. In the figure, channel 100A is sometimes referred to as aone-dimensional channel. A one-dimensional channel has an aperture crosssectional geometry such that a chord of length larger than the diameterof the object to be excluded can be drawn. In addition, thecross-sectional geometry also has a chord of length less than the widthof the object. For example, in the figure, channel 100A has arectangular exterior configuration and uniform wall thickness, thusforming rectangular aperture 105. Aperture 105 has a diameter, asdenoted by dimension 120, and a width, as denoted by dimension 115.Target particle 35 has an average diameter denoted by dimension 37. Asillustrated, width dimension 115 is smaller than dimension 37, and thus,particle 35 is precluded from passing through channel 100A. With targetparticle 35 lodged in the constriction formed by the dimensionaldifference between width dimension 115 and diameter dimension 37, fluidflow in channel 100A is allowed to continue since diameter dimension 120is larger than diameter dimension 37. Fluid bypasses the constrictionand passes around target particle 35 in the presence of continuedapplication of a pressure differential. Pressure build-up on the surfaceof target particle 35 remains relatively low since fluid is allowed tobypass.

FIGS. 1C and 1D illustrate channel geometries in accordance with thepresent subject matter. FIG. 1C illustrates aperture A having crosssectional geometry in the form of a rectangle. Dimension D_(A)represents a diameter of the aperture and dimension W_(A) a width of theaperture. The width represents a minimum distance that can be drawnbetween two parallel supporting lines. The supporting lines are tangentsof the perimeter. The diameter represents a maximum distance that canthat can be drawn between two parallel supporting lines. In the case ofthe rectangular shaped aperture, the supporting lines are co-linear withthe sides of the aperture.

FIG. 1D illustrates arbitrary object O having width denoted by W_(o) anddiameter denoted by D_(O) for a particular cross section, defined as theminimum and the maximum distances, respectively, that can be drawnbetween two parallel support lines. In the case of object O, thediameter supporting lines are denoted D_(SL) and the width supportinglines are W_(SL).

Aperture A will exclude object O when, for a particular cross section,W_(o) is greater than W_(A). Aperture A has cross sectional geometrythat can be described as convex. A shape is convex if it wholly containsthe straight line that joins any two points inside the shape. FIG. 1Gillustrates convex shape P.sub.1 in which line segment L.sub.1 denotes arepresentative line segment having points that lie entirely within theperimeter of shape P.sub.1.

According to the present subject matter, a one-dimensional channel(having an aperture with convex cross sectional geometry) can bedescribed as a channel wherein the aperture width is smaller than thewidth of the object to be excluded and the aperture diameter of thechannel is larger than the diameter of the object as well as larger thanthe width of the aperture. A channel of circular aperture, wherein theaperture diameter is equal to the aperture width, is not considered as aone-dimensional channel.

More generally, the present subject matter includes a channel having anaperture cross sectional geometry such that a chord of length largerthan the diameter of the object to be excluded can be drawn. A chord isa straight line formed between two points on the perimeter of the shape.A chord may cross the perimeter. In other words, a chord may passthrough the region enclosed by the perimeter as well as the regionoutside of the perimeter. FIGS. 1G, 1H and 1J illustrate exemplaryshapes having chords C₁ (perimeter P₁), C₂ (perimeter P₂) and C₃(perimeters P₃ and P₄), respectively.

The shapes illustrated in FIGS. 1H and 1J can be described as non-convexor concave. A shape is concave, or non-convex, if it has the propertythat the line segment connecting any two interior points is not totallycontained in the shape. The shapes illustrated in FIGS. 1H and 1J, forexample, are concave since line segments L₂ and L₃, respectively, can bedrawn such that they cross the boundaries or perimeters of the shape.

According to the present subject matter, a one-dimensional channel(having an aperture with concave cross sectional geometry) can bedescribed as having an aperture which features an inscribed convexpolygon of a width smaller than the width of the object to be excludedand also a convex hull that has a diameter larger than the diameter ofthe object to be excluded. Inscribed means to construct a geometricshape inside another so they have points in common but the inscribedshape does not have any part of it outside the other. A convex hull isthe smallest circumscribed convex shape that encloses an interiornon-convex shape. Circumscribe means to construct a geometric shapeoutside another so they have points in common but the circumscribedshape does not have any part of it inside the other. FIGS. 1E and 1Fillustrate exemplary concave apertures having perimeters P₅ and P₆,respectively, and inscribed polygons ICP₁, and ICP₂, respectively andconvex hulls H₁ and H₂, respectively.

FIGS. 2A and 2B illustrate ways that a channel or an array of channel(s)can be configured in relation to a main flow direction. FIG. 2Aillustrates substrate 200A configured for axial flow wherein the fluidflow enters in the direction denoted by arrow 112A and passes throughthe substrate in the direction denoted by arrow 110. The fluid flow,sometimes referred to as the filtrant, is substantially parallel to, orin the same direction as, the main fluid flow. In one example, substrate200A includes a plurality of channels having one-dimensional geometry.

FIG. 2B illustrates substrate 200B configured for radial or lateral flowwherein the fluid flow enters along axis 112B and traverses thesubstrate in the direction aligned with arrow 110. Substrate 200Bincludes a plurality of one-dimensional channels. In a lateral flowconfiguration, as illustrated in the exemplary apparatus, the filtrantdirection is substantially normal to the main flow. Lateral flow issometimes referred to as cross flow.

The discussion regarding flow is merely exemplary. For instance, thefluid may be moved in a first direction to achieve a particularseparation objective and later moved in a second direction to achieve adifferent objective. By way of example, the fluid flow may be reversedto dislodge the excluded objects from the channels or to isolate aparticular object.

A one-dimensional channel or an array of one-dimensional channels may beemployed within a subunit of a microfluidic device. FIGS. 3A and 3Billustrate an embodiment of one-dimensional channels (formed bysandwiching substrate 200C, which is fabricated with an array ofone-dimensional channels as shown in 200D), between two substratesforming input conduit 80 and output conduit 90. Cut-away view 3B-3B isillustrated in FIG. 3B. Substrate 200C receives the filtrant via inputconduit 80A and discharges the filtrate via output conduit 90A. Fluidflow in input conduit 80A is aligned with the direction of arrow 81, insubstrate 200C aligned with the direction of arrow 201C and in outputconduit 90A aligned with the direction of arrow 91. The structure ofsubstrate 200C, when viewed with a side elevation, includes a pluralityof channels, each of which is shown as a line in substrate 200D. Theflow is in an axial configuration in the example illustrated; howeverthe substrates and the embedded channels can be oriented for a lateralflow configuration. Various embodiments include an arbitrary number ofinput conduits and output conduits. In one example, a one-dimensionalchannel is fabricated directly in the top substrate or bottom substrate,thus eliminating the middle, or sandwiched, substrate.

FIGS. 4A-4G illustrate a variety of aperture cross section geometries,each of which has an elongate portion (many having a polygonal shape)which can be configured in size and shape to capture a target particlein one region and allow passage of fluid in another region. For example,FIG. 4A shows an aperture having a section of a ring or a semicirculararc wherein an elongate portion includes a curved section. As such, atarget particle will be lodged between the two curved surfaces of theaperture and fluid can continue to pass near one or both ends of thearc. FIG. 4B illustrates a diamond shaped aperture wherein the diamondis elongate and has two acute interior angles and two obtuse angles. Thenarrow portion of the aperture (nearest the obtuse angles) will capturethe target particle and fluid will flow in the region of the acuteangles. FIG. 4C illustrates a triangular aperture having one obtuseangle and two acute angles. The narrow portion of the aperture (nearestthe obtuse angle) will capture the target particle and fluid will flowin the region of the acute angles. FIGS. 4D-4G illustrate star-shapedand cross-shaped apertures, each of which can be viewed as having morethan one elongate portion. FIGS. 4D, 4F and 4G can be viewed ascombinations of triangle-shaped or diamond-shaped apertures and FIG. 4Ecan be viewed as a combination of rectangular-shaped openings.

In addition to those shown, other apertures are also contemplatedincluding, for example, a combination of the illustrated shapes. Forexample, an aperture can include an oval or an elongate circular shapeor a segment of a circle having a flat on a side (shaped like the letterD). Furthermore, a particular substrate can have apertures of more thanone shape or more than one particular size.

FIGS. 5A-5D, as well as FIGS. 6A-6D, illustrate an exemplary procedurefor micro fabricating one embodiment of a chip according to the presentsubject matter. FIGS. 5A-5D illustrate production of a molding master ona silicon wafer from which polydimethylsiloxane (PDMS) slabsincorporating an array of one-dimensional channels can be replicated. InFIG. 5A, a negative photoresist is spun onto a silicon wafer. Thephotoresist is baked and then partially exposed to ultraviolet lightthrough a patterned photomask using a mask aligner as shown in FIGS.5B-5C. The portion of the photoresist layer exposed to ultraviolet lightis cross-linked by the radiation, and becomes insoluble in the developersolution. Exposing the photoresist layer to the developer solution, inFIG. 5D, removes uncross-linked photoresist and leaves raised structuresof cross-linked photoresist on the surface of the silicon, essentially anegative relief image of the original photomask. The application ofphotoresist, ultraviolet light exposure, and development in developersolution may be repeated to create multi-level layered structures. Uponcompletion of the desired topography, the resulting master mold ispassivated with fluorosilane to allow a PDMS slab be cast on the mastermold and removed. In an alternative method, positive photoresist layersare used to create positive, relief images. In one method,microstructures are produced directly in silicon or other substratematerials by etching with reactive chemicals in gaseous or liquid phase,by ablating with focused laser beams, or by bombarding with directedcharged particle beams such as ions, electrons or plasma.

FIGS. 6A-D illustrates the production of a PDMS microfluidics deviceusing a master mold. The master mold is produced by photolithography andby additional surface modifications in FIGS. 6A-B. In FIG. 6C, liquidPDMS is poured onto the surface of the master mold and baked to cure theliquid PDMS into a soft, semi-solid slab. In FIG. 6D, the cured PDMSslab is peeled from the master mold, oxidized in an oxygen plasma, andthen bonded against another piece of PDMS slab to form enclosedchannels. In one method, the cured PDMS is peeled from the master moldand bonded to a substrate of material such as glass, quartz, or silicon.In one method, a curable thermoset or photocurable polymer, such asthermoset polyester, polycarbonate, or polymethylmethacrylate, is usedin place of PDMS following a casting-replication process. In one method,the aforementioned microstructures are directly produced in a substrateby chemical etching, laser ablation, or charged particle bombardment andbonded to another substrate to form an enclosed fluidic channel.

FIG. 7 illustrates an exemplary method to replicate microfluidic chipsby injection-molding of thermoplastic materials. Solid plastic pelletsare loaded into the hopper and softened under hydraulic pressure andtemperature. The liquified material is then injected into a master moldwith channel features. Upon cooling the plastic replica solidifies andis removed and bonded to another substrate to form an enclosed fluidicdevice.

FIG. 8 illustrates scanning electron microscope images of exemplarydevice 300. Fluid flow is aligned with the direction illustrated by thearrows and enters at inlet 280 and exits at outlet 290. In particular,enlargement 310 and enlargement 340 each illustrate a plurality of thinchannel walls 345 aligned in a pattern having “corner elements” 350.One-dimensional channels (3 μm aperture width by 20 μm aperture diameterby 20 μm channel axial length) are formed between thin channel walls 345as well as between thin channel walls and corner elements 350. Analytesor biofluids are introduced into device 300 via inlet 280 by thehydrostatic pressure difference due to the difference in liquid heightbetween inlet 280 and outlet 290. The channel surfaces may be modifiedchemically to enhance wetting or to assist in the adsorption of selectcells, particles, or molecules.

Device 300 can be used to separate a subpopulation of cells from wholeblood. Whole blood includes a complex mixture of white blood cells(leucocytes), red blood cells (erythrocytes), platelets, and plasma.Leucocytes are spherical-shaped with diameters ranging from 6 μm to 20μm and are not easily deformed as they contain subcellular compartmentssuch as nucleus and organelles. Erythrocytes, on the other hand, aredisc-shaped fluidic sacs with nominal diameter of 7 μm and height of 2μm. Because erythrocytes contain mostly fluids and have an extremelyflexible cytoskeleton designed to support high degrees of deformation,they can be deformed easily and pass though constrictions even smallerthan their smallest dimension. The use of one-dimensional channelsprovides mechanical exclusion to leucocytes in one dimension and leavesopen areas or bypass regions next to the trapped or captured leucocytessuch that the filtrate or carrier fluid can continue to flow. Anerythrocyte can pass though a one-dimensional channel in a sidewaysmode.

The following exemplary procedure can be used to separate leucocytesfrom whole blood using device 300. A 0.05 ml drop of human whole bloodis added to approximately 0.2 ml isotonic phosphate-buffered saline(PBS) solution containing 1.5 mg/ml of K.sub.3 EDTA as anticoagulant,0.1 M of PBS, and 0.15 M of NaCl. Approximately 1 μL of this mixture ispipetted into the inlet reservoir of the device and additional buffer isadded on top of the fluid reservoir to ensure steady gravity-drivenflow. FIGS. 9A-9F show a sequence of photographs documenting theexclusion of leucocytes from whole blood mixture using device 300. Asthe mixture is passed through device 300, leucocytes (circular objectsat the opening of each channel in the lower center and lower leftcorners of FIGS. 9A-9E) are unable to pass though the one-dimensionalchannels and accumulate near the channel entrances. However, since theaperture diameter is greater than the diameter of a leucocyte, theleucocytes do not block the flow completely and consequently theleukocytes remain undamaged and intact. Erythrocytes, however, are freeto flip or deform while traversing through a one-dimensional channel.The white arrows in FIGS. 9A-9E indicate the trajectory of anerythrocyte moving through a one-dimensional channel by flipping ontoits side. FIG. 9F shows a larger area of device 300 during the cellseparation operation, where leucocytes (white circular objects) can beseen accumulating at more than 90% of channel entrances whileerythrocytes passing freely through the channels. The inlet side ofdevice 300 corresponds to the upper right corner of FIG. 9F.

A leucocyte-separation experiment can be conducted with azero-dimensional channel, as illustrated in FIG. 10, for purposes ofcomparison. FIGS. 10A and 10B show the top and side elevation views of achannel having a square cross section of 2×2 μm and an axial length of20 μm coupled to input and output chambers, each having dimensions of100×13 μm. Identical preparation method is used to prepare the bloodmixture and a syringe is used to deliver the mixture into the inletchamber. FIGS. 10-10H show a sequence of images of a leucocyte (markedby arrows) approaching the channel and completely blocking the channel,and having partially expelled its contents under the applied pressure. Aleucocyte blocking the zero-dimensional constriction forms a completeblockage and the separation function ceases once the fluid flow isprecluded.

A theory as to the physics of channel blockage in relations to the localpressure experienced by cells is illustrated in FIGS. 11A-11C, 12, 13,14A, 14B, 15A and 15B. FIGS. 11A-11C illustrates various scenarios thatcan affect the local pressure experienced by a biological cell inseparation processes. FIG. 11A illustrates the hydrodynamic pressureimparted by a carrier fluid as it flows past a cell. FIG. 11Billustrates the pressure experienced by a cell completely clogging asingle channel. FIG. 11C illustrates the pressure experienced by a cellclogging a channel in the presence of multiple parallel channelsavailable for flow bypass. As discussed herein, a one-dimensionalchannel provides improved performance relative to that of azero-dimensional channel in terms of reducing cell lysing since aone-dimensional channel permits the carrier fluid to flow past thetrapped cells and reduce the pressure escalation associated with thecomplete blockage of a zero-dimensional channel.

FIG. 11A illustrates pressure encountered by a single cell in flow. Thepressure experienced by a cell in a separation environment dependsstrongly on whether the carrier fluid is able to pass around the cell.When a cell is excluded by a one-dimensional channel, the fluid is stillable to flow around the cell, and thus the pressure is essentially thesame as if the cell is a free particle in the flow. The upstream half ofthe cell experiences a higher pressure from the direct impingement offluid, which leads to a local distribution of pressure around the cell(P) given by:

$\begin{matrix}{P = {P_{0} + {\frac{3}{2}\frac{\mu\; V}{R}\cos\;\theta}}} & {{Eq}.\mspace{14mu}(1)}\end{matrix}$where P₀ is the upstream pressure (for convenience, the downstreampressure is assumed to be 0), μ is the viscosity of the carrier fluid, Ris the radius of the cell, and V is the velocity of the fluid. Theangular distribution of the pressure from Eq. (1) is plotted in FIG. 12.FIG. 12 shows pressure distribution from a flow around a sphericalobject where zero degree is defined as the angle opposite of theupstream flow direction.

The maximum pressure difference (ΔP_(max)) between 0° (fluid impinging)and 180° (wake) is given by:

$\begin{matrix}{{\Delta\; P_{\max}} = {3\frac{\mu\; V}{R}}} & {{Eq}.\mspace{14mu}(2)}\end{matrix}$

FIG. 11B illustrates a cell clogging a single channel. In the case wherethe cell completely clogs a zero-dimensional channel (or pore), thecarrier fluid is unable to recombine behind the cell and the pressureacross the cell is simply the same as the externally applied pressuredifferential (i.e. the syringe pump pressure.) The externally appliedpressure differential is always larger than the pressure difference fromthe flow around a cell (e.g. when a cell is trapped by a one-dimensionalchannel), sometimes by several orders of magnitude, because the formeris what is required to drive the flow through the entire filter and thelatter is only a small pressure drop from the fluid wrapping around acell. For a partially clogged channel, some fluid is allowed to pass bythe cell and relieve the pressure difference across the cell. The degreeof relief is related to the unobstructed cross-sectional area availablefor flow. FIG. 13 shows the effect on the cell pressure as a cell coversthe channel opening. In other words, FIG. 13 illustrates cell pressureas a function of the percent of channel area blocked. Data points wereobtained by solving the Navier-Stokes equation numerically for a 5 μm(diameter) by 10 μm (axial length) cylindrical channel partially blockedby a 5 μm (diameter) cell.

FIG. 11C illustrates clogging of one channel in an array of multipleparallel channels. In cases where systems of unclogged parallel channelsare available to allow the carrier fluid to bypass and recombine at theexit side of the filtration area, the pressure experienced by theclogged cell is equivalent to the pressure drop across the uncloggedparallel channels. For one unclogged parallel channel, the pressure dropfrom viscous dissipation (ΔP_(channel)) is given by the Poiseuilleequation:

$\begin{matrix}{{\Delta\; P_{channel}} = \frac{32\;\mu\;{VL}}{D^{2}}} & {{Eq}.\mspace{14mu}(3)}\end{matrix}$where μ is the viscosity of the carrier fluid, D is the diameter of thechannel, L is the axial length of the channel, and V is the velocity ofthe fluid.

For n parallel channels, the pressure drop is reduced by a factor n,since more cross-sectional area is available for flow:

$\begin{matrix}{{\Delta\; P_{n - {channel}}} = \frac{32\;\mu\;{VL}}{{nD}^{2}}} & {{Eq}.\mspace{14mu}(4)}\end{matrix}$

A comparison of this pressure drop to the pressure of a trapped cell ina one-dimensional channel (Eq. (2)) can be made by making simplifyingassumptions. Assume that the axial length of the pore is at least fivetimes the pore diameter (L=SD) and that the diameter is twice the radiusof the channel (D=2R) in Eq. (4). Accordingly, the results are given by:

$\begin{matrix}{{\Delta\; P_{n - {channel}}} = {\frac{32\;\mu\;{V( {5( {2R} )} )}}{{n( {2R} )}^{2}} = {\frac{80}{n}\frac{\mu\; V}{R}}}} & {{Eq}.\mspace{14mu}(5)}\end{matrix}$

A comparison of Eq. (5) with Eq. (2) reveals that n, the number ofunclogged bypass channels, should be approximately 80/3 or 27 in orderto relieve the pressure of one clogged channel to the point ofequivalent to cell trapping by a one-dimensional channel. In otherwords, if more than 4% of the total channels are clogged in a substrateconsisting of purely zero-dimensional channels, then the remainingunclogged channels become less effective than a single one-dimensionalchannel in terms of circumventing the pressure build-up. This poses acapacity issue when devices based on zero-dimensional channels are usedto isolate cells.

Thus, an increased probability of cell lysing arises from having amultitude of zero-dimensional channels. The pressure experienced by thecell is directly related to the tension on the cellular membrane. Inother words, the pressure differential forces the cell to stretch, andwhen the increase in surface area exceeds 2-4% of the original surfacearea, the cell is lysed.

FIG. 14A illustrates a channel of diameter 2R_(ch) completely clogged bya cell with the external diameter R_(out). In the simplified geometryshown in FIG. 14A, the pressure required to sustain curvature to themembrane tension is by Laplace's law:

$\begin{matrix}{{\Delta\; P} = {2{r( {\frac{1}{R_{ch}} - \frac{1}{R_{out}}} )}}} & {{Eq}.\mspace{14mu}(6)}\end{matrix}$where AP is the pressure difference between the outside (P_(out)) andthe inside (P_(in)) of the channel, R_(ch) is the radius of curvatureinside the channel, R_(out) is the radius of curvature of the remainingcell volume outside of the channel, and τ, the membrane tension, isproportional to the surface area change.

FIG. 14B illustrates two channels simultaneously clogged by a cell. Insuch a case, where a cell covers two pores simultaneously, the membranetension is given by adding up the curvatures:

$\begin{matrix}{{\Delta\; P_{2 - {pones}}} = {2{r( {\frac{1}{2r_{ch}} - \frac{1}{R_{out}}} )}}} & {{Eq}.\mspace{14mu}(7)}\end{matrix}$

From Eq. (7), it will be noted that for a constant pressuredifferential, if a cell covers more zero-dimensional channels (pores),the membrane tension τ will increase, and the probability of cell lysingincreases. A difference between a one-dimensional channel and multipleclosely packed zero-dimensional channels (pores, as analyzed above),includes the ability of one-dimensional channels to allow flow to passaround a cell in the same channel, keeping the pressure drop across thecell small, and thus minimizing damage to the trapped cell. Insituations where a one-dimensional channel trap cells in high density,interstitial spaces between closely-packed cells are available for fluidflow, as illustrated in FIG. 15A. The zero-dimensional channelsillustrated in FIG. 15B do not provide an interstitial space.

Separation, isolation, and enrichment of subpopulations of biologicalcells from a complex biofluid mixture may have clinical applications indisease diagnosis. The present subject matter can be used to detectalterations in cell populations as well as the presence of parasites orforeign matters in biofluids to facilitate diagnosis.

In one embodiment, an array of one-dimensional channels can beintegrated into a diagnostic device to facilitate the collection andenrichment of leucocytes and malaria-infected erythrocytes in thediagnosis of malaria. Severe malaria is caused by the parasitePlasmodium falciparum. The parasite invades the erythrocytes in blood,and its maturation process causes the erythrocytes to losedeformability. The physical changes of invaded erythrocytes at thecellular level include the incorporation of knob-associatedhistidine-rich protein (KAHRP) in the cellular membrane, increasedinternal viscosity due to the parasite presence, and a more sphericalsurface-to-volume ratio. Microfluidic observations have provided visualconfirmation that parasitized erythrocytes frequently result incapillary blockage, which has been proposed as the underlyingpathogenesis mechanism.

Current diagnostic protocol for malaria diagnosis includes microscopicexamination of blood smear and the visual identification of malarialparasites. Two microscopy procedures are recommended by the Center forDisease Control (CDC) and the World Health Organization (WHO): in thicksmear preparation, erythrocytes are lysed, and a microscopist visuallycounts the number of parasites against the leucocytes present in 100fields under 100× oil-immersion objective and converts the ratioaccordingly; in thin smear preparation, erythrocytes are not lysed, anda microscopist examines 300 fields under the same magnification andcounts the parasitized erythrocytes among normal erythrocytes.

The present subject matter can be applied to the field of malariadiagnostics. The apparatus can selectively isolate leucocytes as well asparasitized erythrocytes while allowing normal erythrocytes to passthough. The present subject matter also acts as a cell concentrator asthe isolated cells accumulate in an enclosed volume, thus reducing thenumber of fields necessary to achieve the same cell counts compared tothe thick smear protocol. In addition, one example of the presentsubject matter provides a reduced pressure drop across the substrate.

FIGS. 16A-16F illustrate a sequence of photographs showing trapping ofmalaria-infected red blood cells (RBC) and white blood cells (WBC) whileallowing the passage of uninfected red blood cells using an exemplarydevice. One microliter of sample analyte, including malaria-infectedhuman blood diluted with RPMI growth media to ˜10,000 cells/μL ispipetted into the inlet. Additional growth media and dyes may be addedto control the flow rate and improve the visualization of cells andparasites. In one example, the channel surfaces are modified chemicallyto improve trapping of desired cells. The exemplary device is thenplaced onto a Nikon TE300 inverted microscope and inspected under a 100×oil-immersion objective magnification. FIG. 16A shows three parallelone-dimensional channels of aperture dimensions 4 μm width by 16 μmdiameter (channel height) and an axial length of 20 μm, formed betweenfour rectangular walls. The fluid flow is from right to left and drivenby the hydrostatic height difference between the inlet and outletreservoirs (gravity-driven). Immediately adjacent to the channelentrances are one trapped leucocyte (white blood cell) and four infectederythrocytes (RBC). The malaria-infected erythrocytes are spherical andare unable to pass through the one-dimensional channels. Young P.falciparum parasites corresponding to the developmental stage ofring-stage trophozoite are visible in the form of a small white granuleinside two of the infected erythrocytes. FIGS. 16A-16F show theunrestricted movement of an uninfected erythocyte (marked by blackarrow) through a one-dimensional channel by flipping onto its side. Thusthe entrance side of the one-dimensional channels becomes enriched withleucocytes and infected erythrocytes. These cells may be enumerated todocument the parasite concentration and the developmental stages of theparasites may be accurately identified.

In addition to malaria, the present subject matter can be used formonitoring of CD4+ T-lymphocytes (CD4+ T-cells) in HumanImmunodeficiency Virus (HIV) diagnostic and monitoring. The absoluteCD4+ T-lymphocyte count can serve as a criterion to initiateantiretroviral therapy and opportunistic infection prophylaxis inHIV-infected patients. The reduction of CD4+ T-lymphocytes, which is asubpopulation of leucocytes (white blood cells), strongly correlates tothe decline of the immunological defense. Monitoring of CD4+T-lymphocytes (CD4+ T-cells) level every 3-6 months in all HIV-infectedpersons has been recommended by the CDC Public Health Service as a wayto initiate appropriate treatment strategies and to evaluate treatmentefficacy.

In some laboratories, the absolute CD4+ T-cell number is establishedusing the product of three laboratory techniques: the total white bloodcell count, the percentage of white blood cells that are lymphocytes,and the percentage of lymphocytes that are CD4+ T-cells. Single platformflow cytometers such as FACSCount (BD Biosciences) is commerciallyunavailable in some developing countries or as a portable device.

Low cost alternatives for CD4+ T cell monitoring include nonflowbead-based labeling methods with minimal microscope requirements such asmagnetic Dynabeads (DynalBiotech ASA) and latex cytospheres (BeckmanCoulter). Although the measurements from these methods in generalcorrelated well with that from flow cytometry under experienced hands,due to increased manual handling and reading assay, inconsistent resultscan incur, as exemplified by a recent report that number of positivecell can depend on how vigorous the samples were shaken during reagentmixing.

The present subject matter can be used to remove erythrocytes andaccumulate leucocytes prior to appropriate immunophenotyping todistinguish CD4+ T-lymphocytes from other leucocytes. Lymphocytes can bedistinguished from other leucocytes (e.g. monocytes and granulocytes) onthe basis of size, granularity, or morphology and the absolutedistinction of CD4+ T-cell within lymphocytes can be accomplished viaimmunophenotyping. In manual counting methods such as aforementionedDynabeads and Cytospheres, erythrocytes must be lysed with appropriatereagents so leucocytes can be clearly seen, since the ratio of leucocyteto erythrocyte in whole blood is 1:1000. Employing chemical lysingreagents, however, has been known to reduce CD4+ T cell counts by asmuch as 10% when compared to no-lyse methods because lysing agents canlead to destruction of the cell membrane as well as the epitopes forfluorescence labeling. This type of cell count reduction occursnonuniformly among subclasses of leucocytes. In HIV monitoring, wherefalling T-cell count signals the progression of the disease, sucherroneous reduction in absolute count can misguide the physicians ininterpreting the progress of treatment.

In addition to the foregoing disease diagnostic applications,cancer-related rare cells can also be detected using the present subjectmatter. Tumor cells can exfoliate from solid tumors and transportthroughout the body via the blood stream. These circulating tumor cells(CTCs) are present in extremely low concentration in the peripheralblood, estimated to be on the order of one tumor cell per 10⁶ to 10⁷mononuclear cells, which is equivalent to one tumor cell per 0.5 ml to 5ml of peripheral blood. At such low concentration, a sample withestimated 100 million cells must be screened in order to detect at leastone CTC with 99.995% certainty. An automatic digital microscopy (ADM)scanning at a typical speed of 800 cells/second would require 18 hoursto complete a sample that size, and even with an improved opticalsystem, it is estimated that the scanning task would still require aboutone hour with additional manual examination.

The CTCs can be distinguished from normal cells by two physicalcharacteristics: (1) tumor cells preferentially express cytokeratins asintegral components of cytoskeletons, and as such may be distinguishedby means of specific antibodies, and (2) most CTCs have whole cell areas2.8 to 5.7 times larger than normal leucocytes. Current enrichmentmethods of CTCs can be divided into immunological-based approach (e.g.positive and negative immunomagnetic separation) and physical separationmethod (based on filtration using a polycarbonate filter with 8\μmpores). Using filtration, researchers have compared CTCs in patientswith hepatocellular carcinoma before and during surgery, detected CTCsin peripheral blood of breast cancer patients and correlated to thedisease stage, and found that spontaneous circulation of CTCs inperipheral blood is a sign of tumor progression and tumor spread inprimary liver cancer patients, all with high sensitivity.

Since the size difference between cancerous cells and normal cells isconsiderable, the present subject matter can be used to isolate thecirculating tumor cells from whole peripheral blood or spinal fluids andthe system may be configured for inspection under a microscope withoutdisassembling the filtration housing, and without concern of lysing therare cancerous cells.

FIG. 17 includes a photograph of a colon-rectal cancer cell (HT-29 cellline) isolated using the present subject matter. The flow direction inFIG. 17 is from right to left, and the biofluid consists of a dilutemixture of HT-29 cancer cells in McCoy's cell growth medium. The arrowin FIG. 17 marks the trapped cancer cell. Furthermore, additionaldevices or methods for cell separation (e.g. dielectrophoresis,electrophoresis, electrokinetic based separation, magnetically basedseparation, optically based cell sorting) or cell screening (e.g.fluorescence-based screening to identify cancer cells tagged with adye-labeled antibody to cytokeratin from other cells) can be integrateddownstream of this device to confirm the identity of the isolatedcancerous cells.

Additional applications involving separation, concentration andisolation addressed by the present subject matter include fetal cellmonitoring in maternal blood for prenatal diagnostic of geneticdisorders and prion detection. A prion includes a small infectiousproteinaceous particle which resists inactivation by procedures thatmodify nucleic acids. In addition, the present subject matter can beused with fetal cells (fetal cells are larger than maternal cells) andother micro-biological particulates or nano-biological particulates.

As used herein, filtration includes collecting the clarified filtrate aswell as isolating and concentrating solids. Examples of filteringinclude partitioning biological cells and micro-particulates ornano-particulates. Examples of the present subject matter can be usedfor filtering, separating, isolating, concentrating and purifying.

In one example, the present subject matter includes a substrate materialincluding, but not limited to, polymeric materials (polydimethylsiloxane(PDMS), polymethylmethacrylate (PMMA), polyethylene, polyester (PET),polytetrafluoroethylene (PTFE), polycarbonate, polyvinyl chloride,fluoroethylpropylene, lexan, polystyrene, cyclic olefin copolymers,polyurethane, polyestercarbonate, polypropylene, polybutylene,polyacrylate, polycaprolactone, polyketone, polyphthalamide, celluloseacetate, polyacrylonitrile, polysulfone, epoxy polymers, thermoplastics,fluoropolymer, and polyvinylidene fluoride, polyamide, polyimide),inorganic materials (glass, quartz, silicon, GaAs, silicon nitride),fused silica, ceramic, glass (organic), metals and/or other materialsand combinations thereof.

In addition, the substrate can be fabricated of porous membranes, wovenor non-woven fibers (such as cloth or mesh) of wool, metal (e.g.stainless steel or Monel), glass, paper, or synthetic (e.g. nylon,polypropylene, and various polyesters), sintered stainless steel andother metals, and porous inorganic materials such as alumna, silica orcarbon.

The flow can be delivered by, for example, methods and devices thatinduce hydrodynamic fluidic pressure, which includes but is not limitedto those that operate on the basis of mechanical principles (e.g.external syringe pumps, pneumatic membrane pumps, vibrating membranepumps, vacuum devices, centrifugal forces, and capillary action);electrical or magnetic principles (e.g. electroosmotic flow,electrokinetic pumps piezoelectric/ultrasonic pumps, ferrofluidic plugs,electrohydrodynamic pumps, and magnetohydrodynamic pumps); thermodynamicprinciples (e.g. gas bubble generation/phase-change-induced volumeexpansion); surface-wetting principles (e.g. electrowetting, chemically,thermally, and radioactively induced surface-tension gradient).

In addition, fluid drive force can be provided by gravity feed, surfacetension (like capillary action), electrostatic forces (electroosmoticflow), centrifugal flow (substrate disposed on a compact disc androtated), magnetic forces (oscillating ions causes flow),magnetohydrodynamic forces and a vacuum or pressure differential.

Fluid flow control devices, such as those enumerated with regard tomethods and devices for inducing hydrodynamic fluid pressure or fluiddrive force, can be coupled to an input port or an output port of thepresent subject matter. In one example, multiple ports are provided ateither or both of the inlet and outlet and one or more ports are coupledto a fluid flow control device.

The present subject matter can be fabricated by replication or directfabrication. Examples include semiconductor fabrication techniques andmethods including photolithography, growing a crystalline structure, andetching (reactive ion etching and wet etching), laser ablation, replicamolding, injection molding and embossing (application of heat andpressure) and imprinting.

In various examples, the present subject matter is fabricated in theform of a membrane. The membrane can have a uniform thickness or apredetermined thickness gradient. In addition, the uniformity andnumerosity of the pores can be tailored for a particular application.Furthermore, one or more particular coatings can be applied to anexternal or internal surface of the substrate. For example, the channelsurfaces may be modified chemically to increase or decrease the surfaceinteraction with the object or particulate to enhance deviceperformance.

The present subject matter can be integrally fabricated on a chip orconstructed off chip and then assembled onto a chip.

A micro-fabricated or nano-fabricated device and method of the presentsubject matter can be used to separate or filter microscopic ornanoscopic biological objects, such as biological cells, macromolecules,colloidal particles, particulates, or micro-beads.

A plurality of one-dimensional channels can be configured such that thelongitudinal axis of each channel is aligned in parallel for axial flow.A radial configuration of longitudinal axis (converging at a point)yields a cross-flow filter. Other configurations are also contemplated,including, for example, a random arrangement of axes.

According to one example, an apparatus has one or more channels havingan aperture cross sectional geometry such that a chord of length largerthan the diameter of the object to be excluded can be drawn.

In particular, a channel having a convex cross sectional aperture,according to one example of the present subject matter, has an aperturewidth less than the width of the object to be excluded and the aperturediameter is greater than the diameter of the object and also greaterthan the aperture width.

In addition, a channel having a concave cross sectional aperture,according to one example of the present subject matter, has an inscribedconvex polygon of a width and a diameter less than the width of theobject to be excluded and a convex hull that has a diameter greater thanthe diameter of the object to be excluded.

In one example, the object is allowed to move in a direction parallel tothe fluid flow. In other examples, the object is allowed to move in adirection at a particular angle relative to the fluid flow. That anglecan be normal or at any other angle. For example, the object may bedrawn by capillary action or diffusion in a direction that is notaligned with the fluid flow direction.

Cancer Metastases and CTCs.

Embodiments in accordance with the present invention relate to methodsand apparatuses for concentrating and isolating CTCs from body fluids.One embodiment of the present invention includes a micro-fabricated ornano-fabricated device having channels configured for separating andexcluding.

Embodiments in accordance with the present invention can be used toseparate and enrich cancer cells from other cells that may be present ina body fluid. Some biological cells have cellular membranes that are notrigid and are thus highly sensitive to local pressure changes that areoften present in the process of physical exclusion and separation. Forexample, in isolating cells by mechanical exclusion, exposure of thecells to a high-pressure environment can cause cell lysis.

To overcome these challenges in the separation and concentration ofbiological cells and particles, the present subject matter includesdevices and methods for separating and concentrating biological cells orparticles, while reducing the incidence of cell lysis during theseparation, concentration, filtration or isolation procedure.

Embodiments in accordance with the present invention include channelshaving a particular cross-sectional shape for separating cancer cellsfrom biofluids or immiscible objects from a fluid. The cross-sectionalshape of the channels, as employed in embodiments of the present subjectmatter, reduce the hydrodynamic pressure experienced by the cells duringthe separation, isolation and concentration processes and thereforereduce the likelihood of cellular damage.

The present subject matter can be used as diagnostic in trapping rarecirculating tumor cells (CTCs) in cancer monitoring.

Particular embodiments in accordance with the present invention relategenerally to methods and systems to isolate or enrich tumor cells inbiofluids for the diagnosis of cancer, and in particular but not by wayof limitation, to methods and systems that reduce cell lysis or cellularmembrane damage during the isolation or enrichment of tumor cells.

The spreading of cancer cells from the primary tumor is an importantfactor governing the probability of relapse and the survival rate incancer patients. Often it has been argued that metastases rather thanthe primary tumors are responsible for most tumor deaths. Clinically therate of metastases is quite high: about 50% of breast cancer cases thatwere thought to be localized can become metastatic, and even 30% ofpatients with node-negative diagnosis can be expected to develop distantmetastases within five years.

Since cancer cells have the ability to stimulate angiogenesis, as thecells grow unregulated and lose their ability to adhere to each other,they can enter the blood and lymphatic circulation and circulatethroughout the body. These cells are often referred to as CirculatingTumor Cells (CTC), Disseminated Tumor Cells (DTC), Circulating CancerCells (CCC), Circulating Epithelial Cells (CEC), Occult Tumor Cells(OTC), or other similar permutations to indicate the mobile nature ofthese cells, in contrast to the specimens obtained by direct biopsy ofsolid tumors. CTCs have been detected in the blood of patients sufferingfrom all major cancers: prostate, ovarian, breast, gastric, colorectal,renal, lung, pancreatic, and others.

The cancer metastasis mechanism involves multiple processes: first,after the tumor cells are shed from the primary tumor, they follow thecirculation pattern and become arrested at various organs andcapillaries. Once arrested, tumor cells have certain probability ofdeveloping into tumors, depending on the local chemical environments, ormolecular regulations, which may foster CTC accumulation and encouragethe growth of blood vessels to sustain the new tumors.

The physical circulation pattern describes the trajectory of the CTCsand partially explains why, depending on the location of the primarytumor, there are organ-specific patterns in the metastases process. Forexample, CTCs from a breast tumor may follow the blood circulation,which would first travel through heart, then the capillaries of thelungs, and then taken to all other organs via the systemic arterialcirculation.

Clinically it has been observed that breast cancer metastases can occurin bone, liver, brain and lungs. On the other hand, CTCs from a colontumor would be taken first to the capillaries of the liver, through theheart, the capillaries of the lungs, and then the arterial circulation.

This circulation pattern explains well why clinically colorectal cancerstend to spread to liver initially. During the circulation process, CTCsare lodged at various locations because of size restriction: whiletypical capillaries range between 3-8 μm in diameter, CTCs can be aslarge as 20 μm or more. Capillaries in the lungs and the livers are thusparticularly vulnerable to the lodging of CTCs.

In addition to the physical circulation mechanism, some researchers haveargued that chemical “homing” signals such as chemokines and theirreceptors may preferentially attract CTCs to specific organs. Forexample, chemokine receptors CXCR4 and CCR7 are expressed in breastcancer CTCs, while lymph nodes, lung, liver, and bone marrow are knownto be rich in the corresponding ligands CXCL12 and CCL21.

Once CTCs become lodged, their ability to grow into a sustaining tumordepends on local chemical environment. Presence of growth factors suchas parathyroid hormone-related protein and transforming growth factor-β(TGF-β) in the bones, for example, can stimulate growth.

CTC Screening.

CTCs in blood can be used as an additional prognostic factor for thecancer patients. The strength of a CTC test is in its ability to measurehow cancer cells disseminate, before they become tumors large enough tobe detectable by imaging methods. The most important benefit—that ofsaving life—is its role in determining whether a treatment is effective.A cancer patient can also expect significant cost-saving if a CTC testcan eliminate unnecessary (and often not covered by insurance) ComputedTomography (CT) or Positron Emission Tomography (PET) scans, or shortenthe drug treatments that are ineffective.

CTC test is designed to count the number of tumor cells present in ablood sample and allow a pathologist to visually examine the cells.Careful examination of cytomorphology can yield the origin of CTCs andthe developmental stage of cancer. Select clinical literature hasreported that cancer patients with as little as 5 CTCs in their bloodsample have a much lower survival rate. However, forecasting doom is notthe point of a CTC test. It is about learning the result early,interpreting it as a warning sign that the current treatment is noteffective, and promptly switch to a more aggressive treatment strategybefore it is too late.

CTCs are very different from the ordinary blood cells, which include thered blood cells, the white blood cells, and platelets. Platelets are ononly about 2-4 μm in diameter, which is much smaller than other bloodcells. Red blood cells are highly flexible fluidic sacs deliveringnutrients and oxygen to various parts of the body. A red blood cell hasno internal compartments (organelles) inside and carries no DNA. Ahealthy red blood cell looks like a doughnut (“biconcave”) with adiameter of ˜7 μm. White blood cells have internal compartments(organelles), which render them more rigid than red blood cells. Rangingbetween 7-20 μm in diameter, white blood cells exists in severalvarieties, performing different immunity-related functions. On the otherhand, CTCs are substantially larger than these blood cells. CTCs alsohave internal compartments (organelles) that contain DNAs—especially theDNA of the tumor source that the CTCs came from. CTCs may also carry anabundance of abnormal proteins found in the tumor source.

Accurate isolation and detection of CTCs is not a trivial task. CTCs arepresent in extremely low concentration in the peripheral blood,estimated to be on the order of one tumor cell per 10E6 to 10E7mononuclear cells, which is equivalent to one tumor cell per 0.5 ml to 5ml of peripheral blood. At such low concentration, a sample withestimated 100 million cells must be screened in order to detect at leastone CTC with 99.995% certainty. An automatic digital microscopy (ADM)scanning at a typical speed of 800 cells/second would require 18 hoursto complete a sample that size, and even with an improved opticalsystem, it is estimated that the scanning task would still require aboutone hour with additional manual examination.

Biological cells are often sensitive to local pressure change becausecellular membranes are not rigid. In filtering or isolating cells bymechanical exclusion, exposure of the cells to a high pressureenvironment can cause lysing. Lysis refers to the disintegration,rupturing or destruction of a cell. With a cell, such a breakdown iscaused by damage to the plasma (outer) membrane and subsequent loss ofcell contents (cytoplasm, organelles or nucleus) resulting from physicalinsult to the cell. The present subject matter reduces the incidence oflysis in separation, concentration, filtration or isolation.

According to an embodiment of the present invention, a biofluid samplemay be injected into the device described in the present invention,which contains an array of one-dimensional channels to exclude the CTCs.As the biofluid moves through the device, the concentration of CTC isincreased (enriched) as undesirable components of blood, such asplatelets, red blood cells, and white blood cells, preferentially exitthe one-dimensional channels. The exit side of the one-dimensionalchannels may be interconnected to larger flow channels to facilitate theremoval of the undesirable components. A collection area for CTCs may beconstructed by surrounding an open area with one-dimensional channels.

In accordance with one embodiment of the present invention, one or morewalls forming the one-dimensional channels may be made from opticallytransparent material, such that an exemplary device may be placed undera microscope for inspection and viewed through such walls.

In accordance with certain embodiments, prior to the injection thebiofluid may be diluted.

In accordance with certain embodiments, prior to the injection thebiofluid may be stabilized using a fixative to preserve the integrity ofcellular membranes and prolong the shelf-life.

Prior to the injection the biofluid may be treated with cell-permeatingdyes, antibodies attached with fluorescent molecules, or otherfluorescent entities (e.g. quantum dots, nanoparticles, nanobeads,nanocages, or photoactivated fluorophores), fluorescent probes againstcell surface molecules or entities, fluorescent probes againstintracellular molecules or entities, aptamers, or any other fluorescentcompounds, to aid visualization. Alternatively the biofluid may betreated with fluorescent, magnetic, electroactive, or photoactive agentsthat bind preferentially to a specific population or subpopulation ofcells. In a specific example, the agent binding to the target may be anantibody. In accordance with certain embodiments, this pre-collectiontreatment can aid in the identification of particular targets, forexample subpopulation of cancer cells such as putative cancer stemcells.

Further alternatively, following collection of the entities of interestfrom the biofluid, the device may be treated with cell-permeating dyes,antibodies, aptamers, compounds that bind to cell surface molecules orintracellular molecules of interest, as well as fluorescent, magnetic,electroactive, bioactive or photoactive agents that bind preferentiallyto a collected entity of interest. This post-collection treatment canaid in the identification of particular targets, for examplesubpopulation of cancer cells such as putative cancer stem cells.

The channel surfaces may be treated with anticoagulant compounds,compounds that preferentially bind to CTCs, or compounds that preventsticking of cells.

After the injection of CTC-containing biofluids additional dyes ordye-conjugated molecules (such as antibodies, fab fragments, aptamers,ligands, agonists, antagonists, or combinations thereof) may beintroduced into the device to accentuate cellular features and assistthe identification of tumor cells. An example is to use a stain thatreacts with cytokeratins, which are integral components of thecytoskeleton in epithelial cells, to indicate that the cells come fromepithelial sources (most tumors occur in epithelial tissues) rather thanblood sources.

Other dye examples include flurorescein isothiocyanate (FITC)-conjugatedmouse anti-human epithelial antibody (HEA) and phycoerythrin(PE)-conjugated anti-CD45. Other examples of dye-conjugated antibodiesinclude but are not limited to the pan-cytokeratin antibody A45B/B3,AE1/AE3, or CAM5.2 (pan-cytokeratin antibodies that recognizeCytokeratin 8 (CK8), Cytokeratin 18 (CK18), or Cytokeratin 19 (CK19) andones against: breast cancer antigen NY-BR-1 (also known as B726P,ANKRD30A, Ankyrin repeat domain 30A); B305D isoform A or C (B305D-A orB305D-C; also known as antigen B305D); Hermes antigen (also known asAntigen CD44, PGP1); E-cadherin (also known as Uvomorulin, Cadherin-1,CDH1); Carcino-embryonic antigen (CEA; also known as CEACAM5 orCarcino-embryonic antigen-related cell adhesion molecule 5); β-Humanchorionic gonadotophin (β-HCG; also known as CGB, Chronic gonadotrophin,β polypeptide); Cathepsin-D (also known as CTSD); Neuropeptide Yreceptor Y3 (also known as NPY3R; Lipopolysaccharide-associatedprotein3, LAP3, Fusion; Chemokine (CXC motif, receptor 4); CXCR4);Oncogene ERBB1 (also known as c-erbB-1, Epidermal growth factorreceptor, EGFR); Her-2 Neu (also known as c-erbB-2 or ERBB2); GABAreceptor A, pi (π) polypeptide (also known as GABARAP, GABA-A receptor,pi (π) polypeptide (GABA A(π), γ-Aminobutyric acid type A receptor pi(π) subunit), or GABRP); ppGalNac-T(6) (also known asβ-1-4-N-acetyl-galactosaminyl-transferase 6, GalNActransferase 6,GalNAcT6, UDP-N-acetyl-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase 6, or GALNT6); CK7 (also known asCytokeratin 7, Sarcolectin, SCL, Keratin 7, or KRT7); CK8 (also known asCytokeratin 8, Keratin 8, or KRT8); CK18 (also known as Cytokeratin 18,Keratin 18, or KRT18); CK19 (also known as Cytokeratin 19, Keratin 19,or KRT19); CK20 (also known as Cytokeratin 20, Keratin 20, or KRT20);Mage (also known as Melanoma antigen family A subtypes or MAGE-Asubtypes); Mage3 (also known as Melanoma antigen family A 3, or MAGA3);Hepatocyte growth factor receptor (also known as HGFR, Renal cellcarninoma papillary 2, RCCP2, Protooncogene met, or MET); Mucin-1 (alsoknown as MUC1, Carcinoma Antigen 15.3, (CA15.3), Carcinoma Antigen 27.29(CA 27.29); CD227 antigen, Episialin, Epithelial Membrane Antigen (EMA),Polymorphic Epithelial Mucin (PEM), Peanut-reactive urinary mucin (PUM),Tumor-associated glycoprotein 12 (TAG12)); Gross Cystic Disease FluidProtein (also known as GCDFP-15, Prolactin-induced protein, PIP);Urokinase receptor (also known as uPR, CD87 antigen, Plasminogenactivator receptor urokinase-type, PLAUR); PTHrP (parathyroldhormone-related proteins; also known as PTHLH); BS106 (also known asB511S, small breast epithelial mucin, or SBEM); Prostatein-likeLipophilin B (LPB, LPHB; also known as Antigen BU101, Secretoglobinfamily 1D member 2, SCGB1D2); Mammaglobin 2 (MGB2; also known asMammaglobin B, MGBB, Lacryglobin (LGB) Lipophilin C (LPC, LPHC),Secretoglobin family 2A member 1, or SCGB2A1); Mammaglobin (MGB; alsoknown as Mammaglobin 1, MGB1, Mammaglobin A, MGBA, Secretoglobin family2A member 2, or SCGB2A2); Mammary serine protease inhibitor (Maspin,also known as Serine (or cystein) proteinase inhibitor clade B(ovalbumin) member 5, or SERPINB5); Prostate epithelium-specific Etstranscription factor (PDEF; also known as Sterile alpha motif pointeddomain-containing ets transcription factor, or SPDEF); Tumor-associatedcalcium signal transducer 1 (also known as Colorectal carcinoma antigenCO17-1A, Epithelial Glycoprotein 2 (EGP2), Epithelial glycoprotein 40kDa (EGP40), Epithelial Cell Adhesion Molecule (EpCAM),Epithelial-specific antigen (ESA), Gastrointestinal tumor-associatedantigen 733-2 (GA733-2), KS1/4 antigen, Membrane component of chromosome4 surface marker 1 (M4S1), MK-1 antigen, MIC18 antigen, TROP-1 antigen,or TACSTD1); Telomerase reverse transcriptase (also known as Telomerasecatalytic subunit, or TERT); Trefoil Factor 1 (also known as BreastCancer Estrogen-Inducible Sequence, BCEI, Gastrointestinal TrefoilProtein, GTF, pS2 protein, or TFF1); or Trefoil Factor 3 (also known asIntestinal Trefoil Factor, ITF, p1.B; or TFF3).

After the injection of biofluids, additional reagents may be introducedto analyze the cellular contents (DNA or proteins encapsulated by theCTCs) to determine the molecular origins of cancer.

The device according to embodiments of the present invention mayincorporate additional resistive heating elements to perform on-chipcellular assays such as Polymerase Chain Reaction (PCR) or TimePolymerase Chain Reaction (RT-PCR).

The device according to embodiments of the present invention mayincorporate electrodes to manipulate the trajectory of select cells orbiofluid to enhance the separation base on phenomena suchdielectrophoresis or electrowetting.

The device according to embodiments of the present invention may includemagnetic elements to manipulate the trajectory of select cells toimprove separation base on the magnetic susceptibility of the cells orthe micro-magnetic or nano-magnetic particles attached to the cells.

The device according to embodiments of the present invention may includeelectrodes to conduct on-chip chemical assay such as electrophoresis orelectrochromatography.

Electric field or magnetic field may be applied to force a subpopulationof cells to deviate from normal flow pattern and enhance the separationof cells.

Topographical features or relief patterns may be incorporated into thewalls of channels to create pressure gradients which favor concentratingor dispersing of select cells. FIG. 18 illustrates how such reliefpatterns may be incorporated. FIG. 18 illustrates a flow channel withexamples of relief patterns (topographical features) on the bottom wallof the channel. Arrow indicates the direction of flow. Relief 1 shows adiamond-shaped relief at the bottom of the flow channel. Relief 2 showsa triangular shaped relief at the bottom of the flow channel. Thereliefs may span any or combinations of the four walls that form theflow channel. Multiple reliefs or combinations of different shapes ofreliefs may be used. The shapes of the reliefs can be any arbitraryshape, shown as in Relief 3.

Devices in accordance with embodiments of the present invention may befabricated using a variety of methods. FIG. 19 shows a lithographictechnique to directly fabricate features on such a chip using directetching. In a first step a substrate 1900 is provided. In the next stepphotoresist 1092 is applied to the surface of the substrate and thendeveloped to transfer a pattern. Next, portions 1900 a of the substrate1900 exposed by the patterned photoresist, are subjected to etching withgaseous or liquid reactants. The photoresist pattern is then removed,and a transparent substrate 1904 is bonded to the substrate to form anenclosed chip.

Alternatively, FIG. 20 illustrates a production process of an exemplarydevice using polymer resin to replicate from a master mold. First, areplication master 2000 exhibiting a surface relief pattern is formed.Next, resin 2002 is formed over the replication master and then cured.In the next step, the cured resin 2002 a is removed from the master.Finally, the cured resin is bonded to a transparent substrate 2004 toform the enclosed chip.

The device may include flow channels having a dimension that isspecifically limited in size in order to capture and collect cancercells, while allowing fluid to flow around the captured cell. Examplesof such 1D flow channels include but are not limited to those having amaximum single dimension of 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35μm, 40 μm, 45 μm, or 50 μm.

Effusive Filtration

One technique which may particular effective in isolating CTC's withoutdamage is effusive filtration. For purposes of this application,“effusive filtration” refers to configurations where the flowing fluidis actively dispersed or redistributed by the filtration media and/ormorphological features inside the flow channel, such that the localfluid velocity across the filtration media is tuned to minimize thedegree of physical impact of biological cells during the exclusionprocess. The flow may be dispersed in two-dimensions or three-dimensionsto reduce the fluid velocity in the direction of direct impingement ofthe filtration medium.

FIG. 29 shows a simplified plan view of an example of effusivefiltration in accordance with an embodiment of the present invention.Specifically, FIGS. 29A-B show an example of effusive filtration, wherethe filtration medium is an array of 1-D channels, positioned such thatthey converge toward the center of the main flow. FIG. 29A is anillustration and FIG. 29B is a photograph of the streamlines mappedusing fluorescent nanoparticles.

Flow Line A of FIG. 29A illustrates the trajectory of a fluid elementcloser to the conduit wall, traveling to the right, but is forcedthrough a 1-D channel when obstructed by the walls of 1-D channels at anangle, to join the permeate side of the filter. Flow Line B of FIG. 29Aillustrates the trajectory of a fluid element closer to the center ofthe main flow, traveling essentially in parallel to the walls of the 1-Dchannels.

Flow Line C (dashed line) of FIG. 29A illustrates that where the feedchannel is constricted as a result of the convergence, fluid on thepermeate side may overflow back across the filtration medium downstreamfrom where the fluid element initially crossed to join the feed side.The condition for backflow depends on the magnitude of the feedvelocity, the relative fluidic resistances locally across the 1-Dchannels as well as the resistances of the flow conduits on theretentate side and the permeate side.

FIGS. 30A-B illustrate an alternative embodiment of an apparatusconfigured to perform effusive filtration. This embodiment uses effusivefiltration in the form of a bend or curve in the channel to disperse theflow. FIG. 30A is an illustration and FIG. 30B is a photograph of thestreamlines mapped using fluorescent nanoparticles.

Flow Lines A and B illustrate the trajectories of fluid elements in a180° bend, where a portion of the outer wall of the bend is a filtrationmedium consisting of an array of 1-D channels. Flow Line A illustratesthat, because the walls of 1-D channels does not provide sufficientcentripetal force to keep the fluid element in a circular trajectory,the fluid element traverses through the 1-D channels and join thepermeate side. Flow Line B illustrates that fluid elements closer to theinner side of the bend can follow a path parallel to the bend. The ratioof permeate to retentate (flows across the filtration medium and thatremaining the main flow conduit) is highly dependent of the feedvelocity.

In this example of effusive filtration, increasing the feed velocity canresult in an improvement in separation. Increasing the feed velocityresults in higher centrifugal force experienced by objects travelingthrough the bend, thus objects of different sizes or densities may bepreferentially separated. In addition, centrifugal force also results inforcing more portion of the feed through 1-D channels, thus increasingthe permeate and improving the separation.

The placement of 1-D channels need not be regularly spaced. 1-D channelsmay be separated by a large distance (a long wall) which may be used toalter the directions of the fluid. For example, the upstream outerboundary of the bend provided necessary centripetal force to keep thefluid traveling through an arc.

FIG. 31 illustrates another embodiment of a structure for performingeffusive filtration. In this embodiment, the feed is dispersed todistribute the fluid velocity to improve filtration. FIG. 31A is anillustration and FIG. 31B is a photograph of the streamlines mappedusing fluorescent nanoparticles.

Because the flow conduit features an expansion upstream, the fluidvelocity now contains two components: horizontal (along the direction ofinlet feed) and vertical (along the direction of expansion). Flow Line Aillustrates a flow trajectory where it is largely horizontal. Flow LinesC and D illustrate trajectories that have comparable horizontal andvertical components.

Flow Line B (dashed line) illustrates a flow trajectory that is largelyparallel to the expanding wall, and results in a flow largely tangentialto the 1-D channels. Flow line B replaces Flow Line D when the fluidicresistance of the filtration medium ordinarily crossed by Flow Line D islarger than the fluidic resistance of the filtration media crossed byFlow Line A or C. This configuration may arise if the filter sectioncrossed by Flow Line D has 10 nm-wide 1-D channels whereas the sectionscrossed by Lines A and C have 20-um-wide 1-D channels; or if the filtersection crossed by Flow Line D is blocked by cells such that the fluidicresistance increases dynamically.

In the above embodiment, certain apertures are oriented in a directionsubstantially perpendicular to the direction of flow in order toaccomplish axial filtration of the entity. Examples of such aperturesare those located at the far end of the chamber of FIG. 31A, throughwhich flow line A passes. Other apertures in this embodiment areoriented in a direction not substantially perpendicular to the directionof flow in order to accomplish cross-flow filtration of the entity, forexample the apertures in the example of FIG. 30A.

Thus, in a closed filtration system it is possible to exhibitcharacteristics of tangential flow (Flow Line B), as well as axial flow(Flow Line A). However, because of the flow dispersing and thepositioning of the 1-D channels, the fluid velocity impinging on the 1-Dchannels is decreased significantly as compared with the inlet feedvelocity. This lessening of impinging fluid velocity reduces thephysical impact against the walls of the 1D channels, reducing thelikelihood of membrane damage and even cell lysis, thereby offering apossible advantage over conventional techniques.

Additional details regarding the nature and application of effusivefiltration are described in U.S. nonprovisional patent application Ser.No. 11/766,053 filed Jun. 20, 2007 and incorporated by reference hereinfor all purposes.

EXAMPLES Example 1 Breast Cancer

Breast cancer cells (MCF-7, human breast cancer) were cultured inMinimal Essential Media (MEM) with Earl's balanced salt solutionsupplemented with 10% fetal bovine serum and 1% antibiotics(penicillin/streptomycin). Loose tumor cells (10-50 cells) wereextracted from the culture solution using a micropipette (FIGS.21A-21B). The extracted cells were fixed using a formalin solution(5-40% in final volume) for 30 minutes. The nominal diameter of MCF-7cells was 25 μm.

Exemplary devices were fabricated using the approach shown in FIG. 19.The 1-D channels were configured to line the perimeters of a longserpentine channel, with features to disperse the fluid and reduce thevelocity of the flow component directly impinging upon the walls of the1-D channels. The features that affected the impinging velocity includedthe relative flow resistances of the serpentine channel and the permeatechannels (e.g. length, width, bending, corners), and the local flowresistance through 1-D channels (e.g. length, width, angle with respectto flow). The exemplary device was optimized to handle an overallfluidic throughput of 0.01-10 mL/min, while maintaining the impingingvelocity below 3 cm/sec. This impinging velocity represents a 500-foldreduction as compared with the impinging velocity of an axial-flowfilter of identical channel cross-section (10,000 μm²). Specifically,use of a conventional axial-flow filter of comparable channelcross-section instead of the instant effusive filtration device, wouldproduce an impinging velocity of ˜1500 cm/sec, resulting in the lysis offiltered cells.

The fixed tumor cells were injected into an exemplary device using a 1mL syringe, 20 G1½ needle, PE100 tubing, and a syringe pump. Tumor cellswere counted as they entered the active region of an exemplary device(“cells into system”). Tumor cells trapped by the one-dimensionalchannels were subsequently counted (“cells trapped”) and a ratio ofcells trapped versus cells introduced into an exemplary device wascomputed for according to Eq. (8) below.

The results of the recovery testing are shown in FIG. 22A, with breastcancer data indicated in ♦. FIG. 22A illustrates an average recovery 92%of all breast cancer cells introduced into the device. FIG. 22B shows animage of a breast cancer cell (arrow) traveling through an exemplarydevice.% Recovery=[(cells trapped)/(cells into system)]×100  Eq. (8)

To demonstrate the separation of tumor cells from blood cells, a knownquantity of breast cancer cells (MCF-7) were first labeled with DiIlipophilic membrane fluorescent stain and then mixed with 1 mL of 1:10solution of normal whole human blood and 1× phosphate buffer solution(PBS), again using a micropipette as illustrated in FIGS. 21A-B. Thewhole blood was also fixed with 5-40% formalin for 30 minutes. Themixture was then injected into an exemplary device to determine therecovery rate of breast cancer cells at a flow rate between 0.01-5mL/min.

FIG. 23A shows a median recovery of 92% of the tumor cells and thestatistical spread from multiple runs. FIG. 23B is a bright fieldphotograph of cancer cells trapped in the collection region of anexemplary device, where approximately 20 breast cancer cells weretrapped. FIG. 23C shows a fluorescent image of the same area, indicatingthat the cells collected were indeed the breast cancer cells previouslylabeled with DiI.

Example 2 Lung Cancer

Lung cancer cells (A549, human non-small cell lung cancer) were culturedin Dulbecco's Modified Eagles Medium (DMEM) with 4.5 g/L glucose,L-glutamine, and Sodium Pyruvate, 10% fetal bovine serum, 1% MEM aminoacids, 1% Glutamax, and 1% antibiotics (penicillin/streptomycin). Theywere fixed with 5-40% formalin (final volume) for 30 minutes beforeinjection into an exemplary device.

Microfluidic devices having the same architecture and formed in the samemanner as those described in Example 1 above, were used. The injectionprotocol was identical to that described in Example 1. The nominaldiameter of A549 cells was 20 μm.

More than 90% of lung cancer cells were recovered as shown in FIG. 24(indicated with ♦) using an exemplary device, under a volumetric flowrate between 0.01-5 mL/min.

Example 3 Colorectal Cancer

Colorectal cancer cells (HT-29, human colorectal cancer) were culturedin McCoy's 5A Modified Medium with 1.5 mM L-glutamine, 2200 mg SodiumBicarbonate/L, 10% fetal bovine serum, and 1% antibiotics(penicillin/streptomycin). They were fixed with 5-40% formalin (finalvolume) for 30 minutes before injection into an exemplary device. Thenominal diameter of HT-29 cells was 16 μm.

Also more than 90% of colorectal cancer cells were recovered as shown inFIG. 25 (indicated with ♦) using an exemplary device, under a volumetricflow rate between 0.01-5 mL/min.

Example 4

The size distributions of several cultured cancer cell lines weremeasured using an image analysis software. The distributions of the celldiameter are shown in the statistical box plot (FIG. 26): A549(non-small cell lung cancer) and MCF-7 (breast cancer). Both non-smallcell lung cancer and breast cancer cells are mostly between 20-40microns. Table 1 below lists additional measurements of cancer cellscompiled from various literature sources, again showing cell diameterslarger than 25 um in general.

TABLE 1 Cancer cell size measurements compiled from scientificliterature. Cancer Cells Average Diameter (um) Cervical Cancer (HeLa) 28Liver Cancer (HepG2) 28 Liver Cancer (He3B) 32 Prostate Cancer (LNCaP)27

Because red blood cells, which constitute 99.9% of cells in blood, areon the order of 7 microns, simple size exclusion principle can easilyaccomplish 1,000 fold enrichment. For non-small cell lung cancer andbreast cancer, because these cells are significantly larger than evenmost white blood cells, simple size exclusion principle alone canprovide an extremely high degree of enrichment (easily 100,000 foldenrichment).

In this example, cancer cells were labeled with 1 uM lipophilic dye DiI(Ex: 550 nm, Em: 570 nm) prior to spiking into blood sample. Accuratelyknown cultured MCF-7 cells were spiked into 1 mL of whole human bloodusing a microcapillary mounted on a micromanipulator. The number ofbreast cancer cells ranged between 0-60. The blood sample was thendiluted with phosphate buffered saline solution to produce 1:1 solutionof whole blood to 1×PBS. The blood sample was then transferred into asyringe with the outlet connected to the chip using a PE 100 tubing, andthe syringe was mounted onto a syringe pump, operating between 0.01mL/min and 0.5 mL/min. After the injection into the chip was complete,the chip was inspected under a Nikon (TE2000) microscope under 20×magnification to identify the cancer cells isolated. Fluorescentidentification was accomplished using a green laser (Em 532 nm) atapproximately 5 mW.

FIG. 27 shows the number of cancer cells recovered from multiple runs.The curve fit through the recovery data yields a straight line with aslope, which is equivalent to the rate of recovery, of 98%.

Different lines of cancer cells were tested in the chip. Various channelwidths were used to understand the degree of deformability the cancercells may withstand. Again, whole human blood spiked with cancer cellswas used. FIG. 28 shows the cell recovery as a function of the ratio ofcell diameter to the channel width, which is an indirect measurement ofcell deformation should the cell manage to squeeze through the channel.Because the cells are significantly larger than the channel width (2-5times) and the channel height is always larger than the cell diameter,thus forming a 1-D channel, the recovery is consistently above 90%,demonstrating low rates of cell loss.

Embodiments of devices in accordance with the present invention may beused for all screening of circulating tumor cells in any cancer, whichincludes, but is not limited to: oral cancer, nasopharynx cancer, otherpharynx cancers, oesophagus cancer, stomach cancer, colon and rectumcancers, liver cancer, pancreatic cancer, larynx cancer, lung cancer,skin cancer, breast cancer, cervical cancer, corpus uteri cancer,ovarian cancer, prostate cancer, testicular cancer, kidney cancer,bladder cancer, brain cancer, thyroid cancer, non-Hodgkin lymphoma,Hodgkin lymphoma, multiple myeloma, and leukaemia.

Blood is the best place to find CTCs from the point of patient comfortand convenience. However, blood is not where CTCs tend to exist in highconcentration. For a long time, CTCs were detected mainly by looking inthe bone marrow (e.g. spinal aspirate). It is also possible to findexfoliated tumor cells in urine (for prostate cancer), sputum/lung fluid(lung cancer) and other body fluids.

In such cases the task of isolating tumor cells is much simpler;however, because these fluids may not necessarily circulate in the body.However, the cells isolated may not directly indicate the metastaticpotential.

Devices in accordance with embodiment of the present invention may beused for screening tumor cells from any body fluid, including but notlimited to blood, cerebrospinal fluid, synovial fluid, aqueous humour,vitreous humour, amniotic fluid, bile, cerumen (earwax), Cowper's fluid(pre-ejaculatory fluid), Chyle, Chyme, female ejaculate, interstitialfluid, lymph fluid, menses, breast milk, mucus (including snot andphlegm), pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears,urine, vaginal lubrication, and feces.

Because biofluid samples may contain contaminations such as skin debris,clots, and other undesirable components, embodiments of a device inaccordance with the present invention may incorporate additionalpre-enrichment protocols, which include, but are not limited to: filtersto eliminate gross contamination or clots; reagents to lyse blood cells,reagents to stabilize cell integrity, reagents for selective binding ofblood cells or other components in blood to channel surfaces, or tobeads in the channel or device, or to other materials that are packedwithin the channel or device; non-specific absorption of the blood cellsor other components in blood to solid surfaces or particles in thechannel; chromatographic, dielectrophoretic, magnetic, or otherseparation methods to remove undesired components in blood.

Embodiments of a device in accordance with the present invention mayincorporate assay protocols following the cell isolation or enrichment,which includes, but is not limited to: nucleic-acid based methods suchas RNA extraction (with or without amplification), cDNA synthesis(reverse transcription), gene microarrays, DNA extraction, PolymeraseChain Reactions (PCR) (single, nested, quantitative real-time, orlinker-adapter), or DNA-methylation analysis; cytometric methods such asfluorescence in situ hybridization (FISH), laser capturemicrodissection, fluorescence activated cell sorting (FACS), cellculturing, or comparative genomic hybridization (CGH) studies; andchemical assay methods such as electrophoresis, Southern blot analysisor enzyme-linked immunosorbent assay (ELISA).

In one example, the present subject matter includes a substrate materialincluding, but not limited to, polymeric materials (polydimethylsiloxane(PDMS), polymethylmethacrylate (PMMA), polyethylene, polyester (PET),polytetrafluoroethylene (PTFE), polycarbonate, polyvinyl chloride,fluoroethylpropylene, lexan, polystyrene, cyclic olefin copolymers,polyurethane, polyestercarbonate, polypropylene, polybutylene,polyacrylate, polycaprolactone, polyketone, polyphthalamide, celluloseacetate, polyacrylonitrile, polysulfone, epoxy polymers, thermoplastics,fluoropolymer, and polyvinylidene fluoride, polyamide, polyimide),inorganic materials (glass, quartz, silicon, GaAs, silicon nitride),fused silica, ceramic, glass (organic), metals and/or other materialsand combinations thereof.

In addition, the substrate can be fabricated of porous membranes, wovenor non-woven fibers (such as cloth or mesh) of wool, metal (e.g.stainless steel or Monel), glass, paper, or synthetic (e.g. nylon,polypropylene, and various polyesters), sintered stainless steel andother metals, and porous inorganic materials such as alumna, silica orcarbon.

The flow can be delivered by, for example, methods and devices thatinduce hydrodynamic fluidic pressure, which includes but is not limitedto those that operate on the basis of mechanical principles (e.g.external syringe pumps, pneumatic membrane pumps, vibrating membranepumps, vacuum devices, centrifugal forces, and capillary action);electrical or magnetic principles (e.g. electroosmotic flow,electrokinetic pumps piezoelectric/ultrasonic pumps, ferrofluidic plugs,electrohydrodynamic pumps, and magnetohydrodynamic pumps); thermodynamicprinciples (e.g. gas bubble generation/phase-change-induced volumeexpansion); surface-wetting principles (e.g. electrowetting, chemically,thermally, and radioactively induced surface-tension gradient).

In addition, the fluid drive force can be provided by gravity feed,surface tension (like capillary action), electrostatic forces(electroosmotic flow), centrifugal flow (substrate disposed on a compactdisc and rotated), magnetic forces (oscillating ions causes flow),magnetohydrodynamic forces and a vacuum or pressure differential.

Fluid flow control devices, such as those enumerated with regard tomethods and devices for inducing hydrodynamic fluid pressure or fluiddrive force, can be coupled to an input port or an output port of thepresent subject matter. In one example, multiple ports are provided ateither or both of the inlet and outlet and one or more ports are coupledto a fluid flow control device.

It is to be understood that the above description is intended to beillustrative, and not restrictive. For example, the above-describedembodiments (and/or aspects thereof) may be used in combination witheach other. Many other embodiments will be apparent to those of skill inthe art upon reviewing the above description. The scope of the inventionshould, therefore, be determined with reference to the appended claims,along with the full scope of equivalents to which such claims areentitled. In the appended claims, the terms “including” and “in which”are used as the plain-English equivalents of the respective terms“comprising” and “wherein.” Also, in the following claims, the terms“including” and “comprising” are open-ended, that is, a system, device,article, or process that includes elements in addition to those listedafter such a term in a claim are still deemed to fall within the scopeof that claim. Moreover, in the following claims, the terms “first,”“second,” and “third,” etc. are used merely as labels, and are notintended to impose numerical requirements on their objects.

1. A method of isolating cancer cells, the method comprising: passing abiofluid through a flow channel having a bend configured to impart aflow in a direction towards an aperture, wherein the flow channel isenclosed within walls of a microfluidic device and comprises theaperture configured to impede movement of a cancer cell while allowingflow of the biofluid through the aperture, wherein movement of thecancer cell is impeded by a cross-sectional shape of the aperture,wherein the cancer cell covers about 75% or less of a cross-sectionalarea of the aperture, whereby the cross-sectional area of the aperturereduces the fluid pressure applied to the cell.
 2. The method of claim 1further comprising diluting the biofluid prior to passage through theflow channel.
 3. The method of claim 1 further comprising stabilizingthe biofluid with a fixative prior to passage through the flow channel.4. The method of claim 1 further comprising treating the biofluid with amaterial that binds preferentially to the cancer cell to assist invisualization of the cancer cells.
 5. The method of claim 4 wherein thebiofluid is treated with the material having fluorescent, magnetic,electroactive, bioactive, or photoactive properties.
 6. The method ofclaim 1 further comprising visualizing the cancer cell with a microscopethrough a transparent wall of the channel.
 7. The method of claim 1further comprising: collecting the cancer cell; and treating thecollected cancer cell with a material having fluorescent, magnetic,electroactive, bioactive, or photoactive properties.
 8. The method ofclaim 7 wherein the material is configured to identify a subpopulationof cancer cells.
 9. The method of claim 8 subpopulation of cancer cellscomprises a putative cancer stem cell.
 10. A method comprising:collecting one or more cancer cells by flowing a sample of a biofluidthrough a first flow channel having a bend configured to impart a flowin a direction towards an aperture, wherein the first flow channel isenclosed in a microfluidic device and further comprises the aperturedefined by a smallest dimension of a second flow channel, the smallestdimension smaller than a dimension of the one or more cancer cells,wherein the cross-sectional area of the aperture is configured to reducethe fluid pressure applied to the cancer cells, while allowing thecancer cell to cover about 75% or less of the cross-sectional area whilefluid flows around the one or more cancer cells into the second flowchannel; and exposing the cancer cells to a material configured to bindpreferentially thereto; and counting a number of the collected cancercells in order to evaluate a likelihood of metastasis.
 11. The method ofclaim 10 wherein the cancer cells are exposed to the material prior tocollection.
 12. The method of claim 10 wherein the cancer cells areexposed to the material after the collection.
 13. The method of claim 10further comprising determining cancer diagnosis or prognosis based uponthe number of cancer cells collected.
 14. The method of claim 10 furthercomprising determining an effectiveness of cancer treatment based uponthe number of cancer cells collected.